The effect of immunoglobulins and somatic cells on the gravity separation of fat, bacteria, and spores in pasteurized whole milk.

Our objective was to determine the role that immunoglobulins and somatic cells (SC) play in the gravity separation of milk. The experiment comprised 9 treatments: (1) low-temperature pasteurized (LTP; 72°C for 17.31s) whole milk; (2) LTP (72°C for 17.31s) whole milk with added bacteria and spores; (3) recombined LTP (72°C for 17.31s) whole milk with added bacteria and spores; (4) high-temperature pasteurized (HTP; 76°C for 7min) whole milk with added bacteria and spores; (5) HTP (76°C for 7min) whole milk with added bacteria and spores and added colostrum; (6) HTP (76°C for 7min) centrifugally separated, gravity-separated (CS GS) skim milk with HTP (76°C for 7min) low-SC cream with added bacteria and spores; (7) HTP (76°C for 7min) CS GS skim milk with HTP (76°C for 7min) high-SC cream with added bacteria and spores; (8) HTP (76°C for 7min) CS GS skim milk with HTP (76°C for 7min) low-SC cream with added bacteria and spores and added colostrum; and (9) HTP (76°C for 7min) CS GS skim milk with HTP (76°C for 7min) high-SC cream with added bacteria and spores and added colostrum. The milks in the 9 treatments were gravity separated at 4°C for 23h in glass columns. Five fractions were collected by weight from each of the column treatments, starting from the bottom of the glass column: 0 to 5%, 5 to 90%, 90 to 96%, 96 to 98%, and 98 to 100%. The SC, fat, bacteria, and spores were measured in each of the fractions. The experiment was replicated 3 times in different weeks using a different batch of milk and different colostrum. Portions of the same batch of the frozen bacteria and spore solutions were used for all 3 replicates. The presence of both SC and immunoglobulins were necessary for normal gravity separation (i.e., rising to the top) of fat, bacteria, and spores in whole milk. The presence of immunoglobulins alone without SC was not sufficient to cause bacteria, fat, and spores to rise to the top. The interaction between SC and immunoglobulins was necessary to cause aggregates of fat, SC, bacteria, and spores to rise during gravity separation. The SC may provide the buoyancy required for the aggregates to rise to the top due to gas within the SC. More research is needed to understand the mechanism of the gravity-separation process.

Comparison of efficacies of bovine immune colostral antibody and each immunoglobulin class against verotoxin 2, flagellum and somatic cells of Escherichia coli O157:H7 in mice.

PURPOSE: The efficacy of bovine immune colostral (colostral) antibodies against verotoxin (VT) 2, flagellum and somatic cells of Escherichia coli (E. coli) O157:H7 in mice was determined. METHODS: Three major immunoglobulin (Ig) classes were isolated from the colostral antibody against VT2 by affinity chromatography and were used for estimation. Mice inoculated with VT2 were administered each Ig class from the colostral antibody, colostral antibody (colostral whey containing antibody) or serum antibody against VT2 at 1 hour after VT2 inoculation. RESULTS: All control mice (20/20) died after administration of sterilized saline instead of the colostral antibody. The survival rate was 93.3% (14/15) after administration of S-IgA or IgM antibody, or colostral antibody. Survival rates for IgG antibody and serum antibody administration were 80% (12/15) and 60% (9/15), respectively. Serum concentrations of VT2, which was absorbed from the small intestine in mice after administration of VT2 and colostral antibody, were measured by fluorescence enzyme immunoassay (FEIA). Serum concentrations of VT2 after administration of colostral antibody were lower than those after administration of sterilized saline. Mice inoculated with VT2-producing E. coli 157:H7 were administered anti-flagellum or anti-somatic colostral antibodies. Survival rates for E. coli O157:H7-infected mice administered the anti-flagellum and anti-somatic colostral antibodies were 52.4% (11/21) and 22.2% (4/18), respectively. Furthermore, survival rates increased to 89.5% (17/19) with combined administration of anti-flagellum and anti-VT2 colostral antibodies. CONCLUSION: These results suggest that colostral antibodies against VT2, flagellum and somatic cells are effective against E. coli O157:H7 infection.

A novel, cost-effective and efficient chicken egg IgY purification procedure.

Chicken IgY antibodies have been touted to be a superior alternative to mammalian antibodies for use in various immunological, molecular biology and proteomics applications for several reasons. These include, but are not limited to, improved specificity due to maximum phylogenetic distance between host and recipient, cost effectiveness in maintaining commercial numbers of hens, IgY yield and the use of non-invasive methods used to isolate IgY from eggs as opposed to blood. Despite this, the routine use of IgY-based methodologies in the laboratory is not widespread. One reason for this reluctance may be derived from the difficulties and expense of isolating IgY antibodies from egg yolk in sufficient yield, with high purity at a realistic reasonable price. Here, we describe an extremely cost-effective ($5USD per egg), rapid (within 5 h), efficient and optimised technique to isolate high yields (60 mg) of high purity (~80%) chicken IgY from egg yolks using the common plant gums pectin and κ-carrageenan in the presence of calcium chloride to delipidate egg yolk mixtures whilst maintaining IgY in solution and then ammonium sulphate to subsequently precipitate the resulting IgY antibodies to higher purity. Our data demonstrates that this technique results in a high yield and purity of IgY that is comparable (if not superior to) existing commercial IgY isolation kits. The method also allows the isolation of immunologically active IgY which can be used for further downstream immunotechnological processes. Furthermore, it can also be easily implemented in a standard well equipped laboratory, and may be scaled up to commercial quantities (i.e., thousands of eggs).

Factors affecting immunoglobulin concentration in colostrum of healthy Holstein cows immediately after delivery / Fatores que afetam o nível de imunoglobulina no colostro de vacas holandesas sadias imediatamente após o parto

This study analyzed the influence of the number of milkings, number of births, and udder quarter in immunoglobulin (Ig) concentration in the colostrum of healthy Holstein cows. It was collected two samples of colostrum by manual milking, getting the first jets to completion of bacteriological examination and immunoglobulin levels by radial immunodiffusion test in agar gel. Positive samples for bacteriological examination were excluded from this investigation. Medians of immunoglobulin's G, A and M in the colostrum collected before the first and second milking were respectively 9,200 and 6,400mg/dL (p=0.0029); 400 and 200mg/dL (p=0.0018); 800 and 400mg/dL (p=0.0001). Median immunoglobulin concentration in animals that calved once, twice or three times or in cows that calved 4 to 6 times were 6,400; 6,400; 3,200 and 11,200mg/dL IgG; 100, 200, 100 and 800mg/dL IgA ; and 400, 400, 100 and 800mg/dL IgM, respectively. Concentrations of IgG, IgA and IgM were greater in animals that calved more than 4 times (p<0.05). Medians of IgG, IgA and IgM in the right fore quarter (RF), right hind quarter (RH), left fore quarter (LF) and left hind quarter (LH) were, respectively, 7,800; 6,400; 7,800 and 6,400mg/dL; 200, 200, 200 and 200mg/dL; and 400, 400, 400 and 400mg/dL. Ig concentrations in the colostrum of Holstein cows were influenced by the number of milkings after delivery and number of lactations. These variations may be considered risk factors to passive immunity transfer to newborn calves, predisposing them to diseases and causing economic losses to dairy production.

A pesquisa avaliou a influência do número de ordenhas, número de parições e quarto mamário na concentração de imunoglobulinas (Ig) do colostro de vacas hígidas da raça Holandesa. Foram colhidas duas amostras de colostro por ordenha manual, obtendo-se os primeiros jatos para a realização do exame bacteriológico e determinação dos níveis de imunoglobulinas pelo teste de imunodifusão em gel de ágar. As amostras positivas ao exame bacteriológico foram eliminadas desta investigação. Os valores medianos obtidos para a concentração de imunoglobulinas das classes G, A e M do colostro colhido antes da primeira e segunda ordenha foram, respectivamente de 9.200 e 6.400mg/dL (p=0,0029); 400 e 200mg/ dL (p=0,0018); 800 e 400mg/dL (p=0,0001), respectivamente. Os valores medianos da concentração de imunglobulinas, nos animais com apenas 1 parto, 2, 3 ou nas vacas com 4 a 6 partos foram de 6.400, 6.400, 3.200 e 11.200mg/ dL para a IgG; 100, 200, 100 e 800mg/dL para a IgA; e 400, 400, 100 e 800mg/dL para a IgM, respectivamente. As concentrações de IgG, IgA e IgM foram superiores nos animais com mais de 4 partos (p<0,05). Os valores medianos de IgG, IgA e IgM obtidos nos quartos mamários anterior direito (AD), posterior direito (PD), anterior esquerdo (AE) e posterior esquerdo (PE) foram respectivamente 7.800, 6.400, 7.800, 6.400mg/dL; 200, 200, 200, 200mg/dL; e 400, 400, 400 e 400mg/dL, não observando-se diferenças estatísticas (p>0,05) entre os quartos mamários. Os teores de Igs do colostro de vacas Holandesas sofrem influência do número de ordenhas pós-parto e número de lactações. Estas variações podem ser consideradas fatores de risco associados à falha na transferência de imunidade passiva em bezerros neonatos, predispondo-os às doenças e ocasionando perdas produtivas á pecuária.

Isolation of bovine immunoglobulins resistant to peptic digestion: new perspectives in the prevention of failure in passive immunization of neonatal calves.

In calves, neonatal mortality and disease susceptibility are greatly influenced by failure in passive immunization, normally provided by colostrum ingestion just after birth. Formulations projected to replace natural colostrum have not been successful, and one of the possible reasons for such failure is that orally administered Ig are probably digested in the gastrointestinal tract, so they are not absorbed as intact functional molecules. With the aim of finding an adequate colostrum substitute, we used columns of immobilized jacalin, a lectin known by its ability to bind O-linked oligosaccharides, to obtain a colostral Ig population putatively protected against enzymatic cleavage by the presence of sugar chains. Immunoglobulin G1 is a major constituent of colostrum Ig bound to jacalin (JB-Ig). This preparation contains 10% of the total colostral Ig and is typically 3 to 6 times more resistant to pepsin digestion than the Ig contained in the fraction that is not bound to jacalin, which presumably does not contain O-glycans. Mass spectrometry analysis demonstrated that the tryptic peptides obtained from JB-Ig and unbound Ig were similar, indicating that their distinct susceptibility to enzyme hydrolysis was associated with differences in their sugar chains. Therefore, the present research suggests that the bovine colostrum JB-Ig has potential application in the immunotherapy of neonatal calves that have not been supplied with colostrum.

RA175, which is the mouse ortholog of TSLC1, a tumor suppressor gene in human lung cancer, is a cell adhesion molecule.

RA175, a new immunoglobulin superfamily member, is preferentially expressed during differentiation of P19 embryonic carcinoma (EC) cells induced by retinoic acid. In the present study, we isolated mouse RA175 cDNA in its entirety and showed that RA175 is the mouse ortholog of TSLC1, a tumor suppressor gene in human lung cancer. RA175/TSLC1 was localized in the adherent region of human lung squamous carcinoma cells and in the differentiated P19 EC cells. RA175/TSLC1 showed homophilic trans-interaction activity in a Ca(2+)-independent manner. RA175/TSLC1 was preferentially expressed in the polarized cells lining the lumen of developing mouse lung epithelium. This suggests that RA175/TSLC1 is a cell adhesion molecule that is acting as a tumor suppressor gene in the metastasis of lung tumors. RA175/TSLC1 may be necessary for cells to remain tightly associated in the epithelium, thereby suppressing metastasis.

Isolation of immunoglobulin from egg yolk by anionic polysaccharides.

Isolation conditions of immunoglobulin in egg yolk (IgY) were optimized by the addition of various levels of Na-alginate (Alg), lambda-carrageenan (lambda-Cg), Na-carboxymethyl cellulose (CMC), and pectin (PC) to 6-fold diluted yolk. The mixtures were then reacted at pH 4.0-6.0 for 30 min. The optimal isolation conditions of IgY for Alg, lambda-Cg, and CMC were at the 0.1% level and at pH 5.0, while those for PC were at the 0.15% level and at the same pH. The remaining lipid and remaining protein in the supernatants thus obtained was 0.5-3.8% and 10-17%, respectively, and more than 90% of lipoproteins were precipitated. The IgY recovery was determined to be 33-74% by means of single radial immunodiffusion method when IgY was isolated under the optimal conditions. PC showed the best recovery of IgY, while lambda-Cg provided the least. The interactions between polysaccharides and lipoproteins were mainly ionic bonds, hydrophobic interactions, and hydrogen bonds as determined by the addition (0-2.0 M) of NaSCN or urea to the polysaccharide-lipoprotein precipitates.

Isolation of bovine immunoglobulin G subclasses from milk, colostrum, and whey using immobilized egg yolk antibodies.

Immunoaffinity columns were made with specific egg yolk immunoglobulin (Ig) Y against bovine IgG1 and IgG2 and were used to isolate pure IgG1 and IgG2 from Cheddar cheese whey or colostrum. About 10% of the IgY was specific for IgG, and 3% of the IgY was subclass-specific after hyperimmunization of laying hens with either IgG1 or IgG2. Up to 38% of the potential binding capacity of IgY was obtained after immobilization by reductive amination. The IgY columns were stable, and one column could be reused for more than 50 times for over a year with minimal loss in binding capacity. Milk that was free of either IgG subclass was successfully produced by the selective removal of IgG1 or IgG2 subclasses. Double-immunodiffusion analysis confirmed the isolation of subclasses from whey and colostrum and also confirmed that their removal from milk was specific.

[A new method of preparation of bovine colostral immunoglobulins for parenteral administration in calves]. / Nová metoda prípravy hovezích kolostrálních imunoglobulinu k parenterální aplikaci u telat.

A new simple method of preparation of bovine colostral immunoglobulins was described using a single step precipitation of skimmed bovine colostrum with dimethyllaurylbenzylammonium bromide (DMLBAB). This quaternary ammonium compound precipitated simultaneously nearly all colostral proteins lacking antibody activity. Bovine colostrum was collected mostly during of the first 24 hours after calving, at the latest however until 48 hours. Isolation of bovine colostral immunoglobulins proceeded as follows: one volume of skimmed colostrum containing 3-6% of immunoglobulins was slowly precipitated with the same volume of 2% water solution of DMLBAB at pH 7.9-8.1 along with continuous stirring. Turbid mixture was then heated to 43-45 degrees C and subsequently cooled to a room temperature standing overnight. Heavy precipitate sedimented down and supernatant fluid containing purified immunoglobulins was decanted and clarified by filtration. Residual DMLBAB occurring in the filtrate was removed by passage through a column of strongly acid catex, prepared in Na+ form. Purified colostral immunoglobulins were thickened to the required protein concentration by ultrafiltration. Dense retentate was clear and became an amber colour. Average yield of purified colostral immunoglobulins reached 18.8 g/litre of skimmed bovine colostrum (Tab. I). Electrophoretic purity of immunoglobulins fraction amounted to 90-95% (Fig. 1). For parenteral application in calves the above solution of immunoglobulins was subsequently adjusted to 9-11% content of protein, 0.9% of sodium chloride, pH 7.2, stabilized with 2% of aminoacetic acid and conserved with 0.015% of thiomersal. Finally, the preparation was sterilized by filtration, kept its content of immunoglobulins minimally 2 years at the temperature of storage between 2-8 degrees C and remained biologically harmless. Using the method described it was not necessary to remove casein from skimmed bovine colostrum prior to the purification of immunoglobulins. Hence the method provided a significant short cut especially in laboratory as well as pilot scale production of bovine colostral immunoglobulins bringing about a marked economic benefit.

Immunoglobulins of camel (Camelus dromedarius) colostrum.

Immunoglobulins G, M and A were identified in dromedary camel colostra by acid precipitation, gel filtration and fast-protein liquid chromatography (ion exchange). Heavy and light chains were identified by polyacrylamide gel electrophoresis and Western blotting. IgG subclasses (IgG1, IgG2 and IgG3) were isolated by DEAE ion-exchange chromatography and shown to have different electrophoretic mobilities. Cross-reactivity of camel IgA with IgA of other species was determined by enzyme-linked immunosorbent assay (ELISA). Most of the immunoglobulin content was IgG, but a molecule identifiable as IgA was detected and purified. It would appear that in the camel, as in cattle, IgG is the major secretory immunoglobulin of colostrum.

Preventive administration of bovine colostral immunoglobulins for foal diarrhea with rotavirus.

Foal diarrhea due to serotype 3 rotavirus broke out on a foal-raising farm in the years 1987 and 1989. In 1989, all of the foals, regardless of whether or not they suffered from diarrhea, received bovine colostral immunoglobulin (Ig) powder orally for 3 to 5 days during the epidemic. The morbidity of the diarrhea was lower than that observed in 1987, when the Ig powder was not administered to foals. These data suggested that the administration of Ig powder might partially prevent foal diarrhea with rotavirus infection.

Secretory IgA, IgG, and IgM immunoglobulins isolated simultaneously from colostral whey by selective thiophilic adsorption.

In order to evaluate the collective contribution of immunoglobulins to gastrointestinal and immune system development in newborn infants, it would be advantageous to develop a simple and rapid procedure for the selective and quantitative removal of all immunoglobulin classes from colostrum and milk. Toward this end, the major immunoglobulin classes typically present in colostrum (IgM, sIgA, and IgG) have been isolated simultaneously by selective removal from a porcine colostral whey model system using a single-step, preparative scale procedure termed thiophilic adsorption. At neutral pH, the salt-promoted thiophilic affinity of immunoglobulins for the synthetic sulfone-thioether ligands exploits a common (as yet unknown) structural feature of immunoglobulins. The adsorption and recovery procedures operate efficiently under mild buffer conditions permitting the subsequent comparative biochemical analyses of individual immunoglobulin classes for structural and functional heterogeneity. Thus, 1 liter columns of thiophilic adsorbent (T-gel) were employed to obtain pure, structurally intact immunoglobulins from 1 liter batches of porcine colostral whey. The identity and purity of the isolated immunoglobulins were determined under both structure-stabilizing ('native') conditions and denaturing conditions using high-performance size-exclusion chromatography, sucrose density gradient centrifugation, immunodiffusion techniques, SDS-polyacrylamide gradient gel electrophoresis with immunoblotting identification procedures, and two-dimensional electrophoresis. This is the first procedure known to us that allows for the simultaneous one-step isolation (under homologous conditions) of various immunoglobulins with preserved quaternary structure and apparent biological activity.

Selective removal, recovery, and characterization of immunoglobulins from human colostrum.

Investigations into the mechanisms by which Ig in human colostrum influence the development and maturation of both the gastrointestinal and the immune systems of human milk-fed term and preterm infants have been restricted by the paucity of purified human milk Ig. We have developed a simple adsorption procedure for the selective removal and quantitative recovery (95-100%) of intact Ig (secretory IgA, IgG, and IgM) present in human colostral whey. The procedure exploits the rapid, ionic-strength dependent, thiophilic adsorption of Ig during a single pass of colostral whey through a column of beaded agarose with immobilized thioether-sulfone ligands (Anal Biochem 1986;159:217-226). The purity and composition of the adsorbed Ig were verified by SDS-PAGE and sensitive silver-staining protein detection procedures; proteins of approximately 78-80 kD (secretory component), 50-60 kD (heavy chain), and 25 kD (light chain) were observed. The identity, structural integrity, and relative concentrations of the recovered Ig were confirmed by high-performance size-exclusion chromatography, Ouchterlony immunodiffusion, rocket immunoelectrophoresis and ELISA. These results were analyzed and compared with reported values for the concentration of human milk Ig. Thus, the use of thiophilic adsorption appears to facilitate 1) selective removal of Ig from colostrum, enabling the evaluation of remaining components for growth- and immune-potentiating properties, and 2) selective immobilization and recovery of Ig from colostrum under conditions consistent with preserved biologic activity.

Eliciting antibodies in chickens to human dimeric IgA removal of factors from human colostrum depressing anti IgA antibody production.

Contrary to expectation chickens did not readily elicit antibodies to IgA dimers when untreated human colostrum was used as antigen. When colostrum was fractionated by means of a column of 8% granulated agarose equilibrated with 10mM phosphate buffer, pH 7.2, a major and a minor fraction were obtained. The major or "1st fraction" consisted of two components with sedimentation coefficients of 10.9 S and 14.1 S, respectively. The minor or "2nd fraction" consisted of components of S values ranging from 2 to 6 and small amounts of 10.9 and 14.1 S material. When chickens were immunized with the "1st fraction" antibodies to dimeric IgA were produced. When the "1st and 2nd fractions" of the column were remixed and used for immunization of chickens, the immune response was poor as when the chickens were injected with untreated colostrum. An immuno-depressing agent in colostrum was indicated. When rabbits were immunized with clarified human colostrum, antibodies against five antigens were elicited, one of the antigens being dimeric IgA. The purified agent suppressed antibody formation in chickens against the haemocyanin of Jasus lalandii. The "activity" is therefore not specific for IgA and the remaining four antigens in human colostrum. The purified component is a glyco-protein with a hexose content in excess of 10%. The derivatized sugars prepared from it were shown by gas liquid chromatography to be an equimolar mixture of galactose, mannose and fucose. The molecular weight (Mr) of the purified component was found to be 72,000 by sedimentation and diffusion and 80,000 by SDS page using Mr reference standards. The properties of the immuno-suppressor strongly suggest that it is the secretory piece of dimeric IgA.

[Isolation of monospecific antisera to guinea pig immunoglobulins]. / Poluchenie monospetsificheskikh antisyvorotok k immunoglobulinam morskoi svinki.

The results of the production and analysis of monospecific rabbit antisera to guinea pig IgG1, IgG2, IgA and IgM are presented. Isolated immunoglobulins of different isotypes, as well as immune precipitates obtained by immunoelectrophoresis, were used for immunization. After adsorption antisera of each type there formed one precipitation line with guinea pig serum in immunoelectrophoresis, thus indicating that they contained antibodies to immunoglobulins of the definite isotype.

Isolation and characterization of root nodule proteins from lupin.

A group of root nodule-specific plant proteins (nodulins) has been isolated from yellow lupin (Lupinus luteus) by immunoaffinity chromatography. The cytoplasmic nodule protein extract was initially enriched in nodulins on a column with immobilized IgG fraction. It was then purified by chromatography on Sepharose 4B - bound IgG against uninfected root proteins and finally on Sepharose 4B - bound IgG against Rhizobium lupini proteins. Rocket immunoelectrophoresis showed that the nodulin preparation did not react with antibodies against root or bacterial proteins. SDS gel electrophoresis of lupin nodulins revealed at least 23 polypeptides ranging in Mr, from 7,000 to 70,000, probably representing protein subunits.

Lymphoblastoid cell-produced immunoglobulins: preparative purification from culture medium by hydroxylapatite chromatography.

Hydroxylapatite (HA) is a chromatography matrix capable of separating nucleic acid as well as protein species. HA adsorbs biomolecules as a function of the extent and distribution of their surface charge. HA has been evaluated for its ability to differentially retain immunoglobulin molecules, which are planar relative to the generally globular serum proteins, particularly albumin, contained in tissue culture medium. HA chromatography provides a single-step method to purify and concentrate immunoglobulin proteins secreted by lymphoblastoid cells into culture medium from the vast excess of serum proteins used to supplement the medium. A human lambda light-chain protein was recovered in 5% of the applied volume of medium, and was separated from 95% of the total protein. More than 75% of a hybridoma-produced complete immunoglobulin was recovered essentially free of serum protein contamination. HA appears to provide a valuable alternative chromatographic medium for the purification of immunoglobulin proteins secreted by lymphoblastoid cells.

[Antilymphocytic immunoglobulin with an associated antibacterial orientation. I. The production and experimental laboratory study of the preparation]. / Antilimfotsitarnyi immunoglobulin s assotsiirovannoi antibakterial'noi napralennost'iu. Soobshchenie I. Poluchenie i laboratorno-éksperimental'noe issledovanie preparata.

Immunoglobulin was obtained from rabbit sera following immunization of the animals with a complex antigen composed of a suspension of human thymocytes and killed microbe polyvaccine. The preparation possessed not only antilymphocytic activity in vitro, but also protected the animals after their artificial infection.